Generation of Plants with Altered Oil Content

ABSTRACT

The present invention is directed to plants that display an altered oil content phenotype due to altered expression of a HIO1002 nucleic acid. The invention is further directed to methods of generating plants with an altered oil content phenotype.

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application60/575,202 filed May 28, 2004, the contents of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

The ability to manipulate the composition of crop seeds, particularlythe content and composition of seed oils, has important applications inthe agricultural industries, relating both to processed food oils and tooils for animal feeding. Seeds of agricultural crops contain a varietyof valuable constituents, including oil, protein and starch. Industrialprocessing can separate some or all of these constituents for individualsale in specific applications. For instance, nearly 60% of the U.S.soybean crop is crushed by the soy processing industry. Soy processingyields purified oil, which is sold at high value, while the remainder issold principally for lower value livestock feed (US Soybean Board, 2001Soy Stats). Canola seed is crushed to produce oil and the co-productcanola meal (Canola Council of Canada). Nearly 20% of the 1999/2000 UScorn crop was industrially refined, primarily for production of starch,ethanol and oil (Corn Refiners Association). Thus, it is often desirableto maximize oil content of seeds. For instance, for processed oilseedssuch as soy and canola, increasing the absolute oil content of the seedwill increase the value of such grains. For processed corn it may bedesired to either increase or decrease oil content, depending onutilization of other major constituents. Decreasing oil may improve thequality of isolated starch by reducing undesired flavors associated withoil oxidation. Alternatively, in ethanol production, where flavor isunimportant, increasing oil content may increase overall value. In manyfed grains, such as corn and wheat, it is desirable to increase seed oilcontent, because oil has higher energy content than other seedconstituents such as carbohydrate. Oilseed processing, like most grainprocessing businesses, is a capital-intensive business; thus smallshifts in the distribution of products from the low valued components tothe high value oil component can have substantial economic impacts forgrain processors.

Biotechnological manipulation of oils can provide compositionalalteration and improvement of oil yield. Compositional alterationsinclude high oleic soybean and corn oil (U.S. Pat. Nos. 6,229,033 and6,248,939), and laurate-containing seeds (U.S. Pat. No. 5,639,790),among others. Work in compositional alteration has predominantly focusedon processed oilseeds but has been readily extendable to non-oilseedcrops, including corn. While there is considerable interest inincreasing oil content, the only currently practiced biotechnology inthis area is High-Oil Corn (HOC) technology (DuPont, U.S. Pat. No.5,704,160). HOC employs high oil pollinators developed by classicalselection breeding along with elite (male-sterile) hybrid females in aproduction system referred to as TopCross. The TopCross High Oil systemraises harvested grain oil content in maize from about 3.5% to about 7%,improving the energy content of the grain.

While it has been fruitful, the HOC production system has inherentlimitations. First, the system of having a low percentage of pollinatorsresponsible for an entire field's seed set contains inherent risks,particularly in drought years. Second, oil contents in current HOCfields have plateaued at about 9% oil. Finally, high-oil corn is notprimarily a biochemical change, but rather an anatomical mutant(increased embryo size) that has the indirect result of increasing oilcontent. For these reasons, an alternative high oil strategy,particularly one that derives from an altered biochemical output, wouldbe especially valuable.

The most obvious target crops for the processed oil market are soy andrapeseed, and a large body of commercial work (e.g., U.S. Pat. No.5,952,544; PCT application WO9411516) demonstrates that Arabidopsis isan excellent model for oil metabolism in these crops. Biochemicalscreens of seed oil composition have identified Arabidopsis genes formany critical biosynthetic enzymes and have led to identification ofagronomically important gene orthologs. For instance, screens usingchemically mutagenized populations have identified lipid mutants whoseseeds display altered fatty acid composition (Lemieux et al., 1990;James and Dooner, 1990). T-DNA mutagenesis screens (Feldmann et al.,1989) that detected altered fatty acid composition identified the omega3 desaturase (FAD3) and delta-12 desaturase (FAD2) genes (U.S. Pat. No.5,952,544; Yadav et al., 1993; Okuley et al., 1994). A screen whichfocused on oil content rather than oil quality, analyzedchemically-induced mutants for wrinkled seeds or altered seed density,from which altered seed oil content was inferred (Focks and Benning,1998). Another screen, designed to identify enzymes involved inproduction of very long chain fatty acids, identified a mutation in thegene encoding a diacylglycerol acyltransferase (DGAT) as beingresponsible for reduced triacyl glycerol accumulation in seeds (KatavicV et al, 1995). It was further shown that seed-specific over-expressionof the DGAT cDNA was associated with increased seed oil content (Jako etal., 2001).

Activation tagging in plants refers to a method of generating randommutations by insertion of a heterologous nucleic acid constructcomprising regulatory sequences (e.g., an enhancer) into a plant genome.The regulatory sequences can act to enhance transcription of one or morenative plant genes; accordingly, activation tagging is a fruitful methodfor generating gain-of-function, generally dominant mutants (see, e.g.,Hayashi et al., 1992; Weigel D et al. 2000). The inserted constructprovides a molecular tag for rapid identification of the native plantwhose mis-expression causes the mutant phenotype. Activation tagging mayalso cause loss-of-function phenotypes. The insertion may result indisruption of a native plant gene, in which case the phenotype isgenerally recessive.

Activation tagging has been used in various species, including tobaccoand Arabidopsis, to identify many different kinds of mutant phenotypesand the genes associated with these phenotypes (Wilson et al., 1996,Schaffer et al., 1998, Fridborg et al., 1999; Kardailsky et al., 1999;Christensen S et al., 1998).

SUMMARY OF THE INVENTION

The invention provides a transgenic plant having a high oil phenotype.The transgenic plant comprises a transformation vector comprising anucleotide sequence that encodes or is complementary to a sequence thatencodes a HIO1002 polypeptide. In preferred embodiments, the transgenicplant is selected from the group consisting of rapeseed, soy, corn,sunflower, cotton, cocoa, safflower, oil palm, coconut palm, flax,castor and peanut. The invention further provides a method of producingoil comprising growing the transgenic plant and recovering oil from saidplant.

The invention also provides a transgenic plant cell having a high oilphenotype. The transgenic plant cell comprises a transformation vectorcomprising a nucleotide sequence that encodes or is complementary to asequence that encodes a High Oil (hereinafter “HIO1002”) polypeptide. Inpreferred embodiments, the transgenic plant cell is selected from thegroup consisting of rapeseed, soy, corn, sunflower, cotton, cocoa,safflower, oil palm, coconut palm, flax, castor and peanut. In otherembodiments, the plant cell is a seed, pollen, propagule, or embryocell. The invention further provides feed, meal, grain, food, or seedcomprising a nucleic acid sequence that encodes a HIO1002 polypeptide.The invention also provides feed, meal, grain, food, or seed comprisingthe HIO1002 polypeptide, or an ortholog thereof.

The transgenic plant of the invention is produced by a method thatcomprises introducing into progenitor cells of the plant a planttransformation vector comprising a nucleotide sequence that encodes oris complementary to a sequence that encodes a HIO1002 polypeptide, andgrowing the transformed progenitor cells to produce a transgenic plant,wherein the HIO1002 polynucleotide sequence is expressed causing thehigh oil phenotype. The invention further provides plant cells obtainedfrom said transgenic plant.

The present invention also provides a container of over about 10,000,more preferably about 20,000, and even more preferably about 40,000seeds where over about 10%, more preferably about 25%, more preferablyabout 50%, and even more preferably about 75% or more preferably about90% of the seeds are seeds derived from a plant of the presentinvention.

The present invention also provides a container of over about 10 kg,more preferably about 25 kg, and even more preferably about 50 kg seedswhere over about 10%, more preferably about 25%, more preferably about50%, and even more preferably about 75% or more preferably about 90% ofthe seeds are seeds derived from a plant of the present invention.

Any of the plants or parts thereof of the present invention may beprocessed to produce a feed, meal, or oil preparation. A particularlypreferred plant part for this purpose is a seed. In a preferredembodiment the feed, meal, or oil preparation is designed for ruminantanimals. Methods to produce feed, meal, and oil preparations are knownin the art. See, for example, U.S. Pat. Nos. 4,957,748; 5,100,679;5,219,596; 5,936,069; 6,005,076; 6,146,669; and 6,156,227. The meal ofthe present invention may be blended with other meals. In a preferredembodiment, the meal produced from plants of the present invention orgenerated by a method of the present invention constitutes greater thanabout 0.5%, about 1%, about 5%, about 10%, about 25%, about 50%, about75%, or about 90% by volume or weight of the meal component of anyproduct. In another embodiment, the meal preparation may be blended andcan constitute greater than about 10%, about 25%, about 35%, about 50%,or about 75% of the blend by volume.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless otherwise indicated, all technical and scientific terms usedherein have the same meaning as they would to one skilled in the art ofthe present invention. Practitioners are particularly directed toSambrook et al., 1989, and Ausubel F M et al., 1993, for definitions andterms of the art. It is to be understood that this invention is notlimited to the particular methodology, protocols, and reagentsdescribed, as these may vary.

As used herein, the term “vector” refers to a nucleic acid constructdesigned for transfer between different host cells. An “expressionvector” refers to a vector that has the ability to incorporate andexpress heterologous DNA fragments in a foreign cell. Many prokaryoticand eukaryotic expression vectors are commercially available. Selectionof appropriate expression vectors is within the knowledge of thosehaving skill in the art.

A “heterologous” nucleic acid construct or sequence has a portion of thesequence that is not native to the plant cell in which it is expressed.Heterologous, with respect to a control sequence refers to a controlsequence (i.e. promoter or enhancer) that does not function in nature toregulate the same gene the expression of which it is currentlyregulating. Generally, heterologous nucleic acid sequences are notendogenous to the cell or part of the genome in which they are present,and have been added to the cell, by infection, transfection,microinjection, electroporation, or the like. A “heterologous” nucleicacid construct may contain a control sequence/DNA coding sequencecombination that is the same as, or different from a controlsequence/DNA coding sequence combination found in the native plant.

As used herein, the term “gene” means the segment of DNA involved inproducing a polypeptide chain, which may or may not include regionspreceding and following the coding region, e.g. 5′ untranslated (5′ UTR)or “leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons) and non-transcribed regulatory sequence.

As used herein, “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid sequence or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all as a result of deliberate humanintervention.

As used herein, the term “gene expression” refers to the process bywhich a polypeptide is produced based on the nucleic acid sequence of agene. The process includes both transcription and translation;accordingly, “expression” may refer to either a polynucleotide orpolypeptide sequence, or both. Sometimes, expression of a polynucleotidesequence will not lead to protein translation. “Over-expression” refersto increased expression of a polynucleotide and/or polypeptide sequencerelative to its expression in a wild-type (or other reference [e.g.,non-transgenic]) plant and may relate to a naturally-occurring ornon-naturally occurring sequence. “Ectopic expression” refers toexpression at a time, place, and/or increased level that does notnaturally occur in the non-altered or wild-type plant.“Under-expression” refers to decreased expression of a polynucleotideand/or polypeptide sequence, generally of an endogenous gene, relativeto its expression in a wild-type plant. The terms “mis-expression” and“altered expression” encompass over-expression, under-expression, andectopic expression.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or “transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell where the nucleicacid sequence may be incorporated into the genome of the cell (forexample, chromosome, plasmid, plastid, or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed (for example,transfected mRNA).

As used herein, a “plant cell” refers to any cell derived from a plant,including cells from undifferentiated tissue (e.g., callus) as well asplant seeds, pollen, propagules and embryos.

As used herein, the terms “native” and “wild-type” relative to a givenplant trait or phenotype refers to the form in which that trait orphenotype is found in the same variety of plant in nature.

As used herein, the term “modified” regarding a plant trait, refers to achange in the phenotype of a transgenic plant relative to the similarnon-transgenic plant. An “interesting phenotype (trait)” with referenceto a transgenic plant refers to an observable or measurable phenotypedemonstrated by a T1 and/or subsequent generation plant, which is notdisplayed by the corresponding non-transgenic (i.e., a genotypicallysimilar plant that has been raised or assayed under similar conditions).An interesting phenotype may represent an improvement in the plant ormay provide a means to produce improvements in other plants. An“improvement” is a feature that may enhance the utility of a plantspecies or variety by providing the plant with a unique and/or novelquality. An “altered oil content phenotype” refers to measurablephenotype of a genetically modified plant, where the plant displays astatistically significant increase or decrease in overall oil content(i.e., the percentage of seed mass that is oil), as compared to thesimilar, but non-modified plant. A high oil phenotype refers to anincrease in overall oil content.

As used herein, a “mutant” polynucleotide sequence or gene differs fromthe corresponding wild type polynucleotide sequence or gene either interms of sequence or expression, where the difference contributes to amodified plant phenotype or trait. Relative to a plant or plant line,the term “mutant” refers to a plant or plant line which has a modifiedplant phenotype or trait, where the modified phenotype or trait isassociated with the modified expression of a wild type polynucleotidesequence or gene.

As used herein, the term “T1” refers to the generation of plants fromthe seed of T0 plants. The T1 generation is the first set of transformedplants that can be selected by application of a selection agent, e.g.,an antibiotic or herbicide, for which the transgenic plant contains thecorresponding resistance gene. The term “T2” refers to the generation ofplants by self-fertilization of the flowers of T1 plants, previouslyselected as being transgenic. T3 plants are generated from T2 plants,etc. As used herein, the “direct progeny” of a given plant derives fromthe seed (or, sometimes, other tissue) of that plant and is in theimmediately subsequent generation; for instance, for a given lineage, aT2 plant is the direct progeny of a T1 plant. The “indirect progeny” ofa given plant derives from the seed (or other tissue) of the directprogeny of that plant, or from the seed (or other tissue) of subsequentgenerations in that lineage; for instance, a T3 plant is the indirectprogeny of a T1 plant.

As used herein, the term “plant part” includes any plant organ ortissue, including, without limitation, seeds, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores. Plant cells can be obtained fromany plant organ or tissue and cultures prepared therefrom. The class ofplants which can be used in the methods of the present invention isgenerally as broad as the class of higher plants amenable totransformation techniques, including both monocotyledenous anddicotyledenous plants.

As used herein, “transgenic plant” includes a plant that compriseswithin its genome a heterologous polynucleotide. The heterologouspolynucleotide can be either stably integrated into the genome, or canbe extra-chromosomal. Preferably, the polynucleotide of the presentinvention is stably integrated into the genome such that thepolynucleotide is passed on to successive generations. A plant cell,tissue, organ, or plant into which the heterologous polynucleotides havebeen introduced is considered “transformed”, “transfected”, or“transgenic”. Direct and indirect progeny of transformed plants or plantcells that also contain the heterologous polynucleotide are alsoconsidered transgenic.

Various methods for the introduction of a desired polynucleotidesequence encoding the desired protein into plant cells are available andknown to those of skill in the art and include, but are not limited to:(1) physical methods such as microinjection, electroporation, andmicroprojectile mediated delivery (biolistics or gene gun technology);(2) virus mediated delivery methods; and (3) Agrobacterium-mediatedtransformation methods.

The most commonly used methods for transformation of plant cells are theAgrobacterium-mediated DNA transfer process and the biolistics ormicroprojectile bombardment mediated process (i.e., the gene gun).Typically, nuclear transformation is desired but where it is desirableto specifically transform plastids, such as chloroplasts or amyloplasts,plant plastids may be transformed utilizing a microprojectile-mediateddelivery of the desired polynucleotide.

Agrobacterium-mediated transformation is achieved through the use of agenetically engineered soil bacterium belonging to the genusAgrobacterium. A number of wild-type and disarmed strains ofAgrobacterium tumefaciens and Agrobacterium rhizogenes harboring Ti orRi plasmids can be used for gene transfer into plants. Gene transfer isdone via the transfer of a specific DNA known as “T-DNA” that can begenetically engineered to carry any desired piece of DNA into many plantspecies.

Agrobacterium-mediated genetic transformation of plants involves severalsteps. The first step, in which the virulent Agrobacterium and plantcells are first brought into contact with each other, is generallycalled “inoculation”. Following the inoculation, the Agrobacterium andplant cells/tissues are permitted to be grown together for a period ofseveral hours to several days or more under conditions suitable forgrowth and T-DNA transfer. This step is termed “co-culture”. Followingco-culture and T-DNA delivery, the plant cells are treated withbactericidal or bacteriostatic agents to kill the Agrobacteriumremaining in contact with the explant and/or in the vessel containingthe explant. If this is done in the absence of any selective agents topromote preferential growth of transgenic versus non-transgenic plantcells, then this is typically referred to as the “delay” step. If donein the presence of selective pressure favoring transgenic plant cells,then it is referred to as a “selection” step. When a “delay” is used, itis typically followed by one or more “selection” steps.

With respect to microprojectile bombardment (U.S. Pat. No. 5,550,318(Adams et al.); U.S. Pat. No. 5,538,880 (Lundquist et. al.), U.S. Pat.No. 5,610,042 (Chang et al.); and PCT Publication WO 95/06128 (Adams etal.); each of which is specifically incorporated herein by reference inits entirety), particles are coated with nucleic acids and deliveredinto cells by a propelling force. Exemplary particles include thosecomprised of tungsten, platinum, and preferably, gold.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System(BioRad, Hercules, Calif.), which can be used to propel particles coatedwith DNA or cells through a screen, such as a stainless steel or Nytexscreen, onto a filter surface covered with monocot plant cells culturedin suspension.

Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species. Examples of species thathave been transformed by microprojectile bombardment include monocotspecies such as maize (International Publication No. WO 95/06128 (Adamset al.)), barley, wheat (U.S. Pat. No. 5,563,055 (Townsend et al)incorporated herein by reference in its entirety), rice, oat, rye,sugarcane, and sorghum; as well as a number of dicots including tobacco,soybean (U.S. Pat. No. 5,322,783 (Tomes et al.), incorporated herein byreference in its entirety), sunflower, peanut, cotton, tomato, andlegumes in general (U.S. Pat. No. 5,563,055 (Townsend et al.)incorporated herein by reference in its entirety).

To select or score for transformed plant cells regardless oftransformation methodology, the DNA introduced into the cell contains agene that functions in a regenerable plant tissue to produce a compoundthat confers upon the plant tissue resistance to an otherwise toxiccompound. Genes of interest for use as a selectable, screenable, orscorable marker would include but are not limited to GUS, greenfluorescent protein (GFP), luciferase (LUX), antibiotic or herbicidetolerance genes. Examples of antibiotic resistance genes include thepenicillins, kanamycin (and neomycin, G418, bleomycin); methotrexate(and trimethoprim); chloramphenicol; kanamycin and tetracycline.Polynucleotide molecules encoding proteins involved in herbicidetolerance are known in the art, and include, but are not limited to apolynucleotide molecule encoding 5-enolpyruvylshikimate-3-phosphatesynthase (EPSPS) described in U.S. Pat. No. 5,627,061 (Barry, et al.),U.S. Pat. No. 5,633,435 (Barry, et al.), and U.S. Pat. No. 6,040,497(Spencer, et al.) and aroA described in U.S. Pat. No. 5,094,945 (Comai)for glyphosate tolerance; a polynucleotide molecule encoding bromoxynilnitrilase (Bxn) described in U.S. Pat. No. 4,810,648 (Duerrschnabel, etal.) for Bromoxynil tolerance; a polynucleotide molecule encodingphytoene desaturase (crtI) described in Misawa et al, (1993) Plant J.4:833-840 and Misawa et al, (1994) Plant J. 6:481-489 for norflurazontolerance; a polynucleotide molecule encoding acetohydroxyacid synthase(AHAS, aka ALS) described in Sathasiivan et al. (1990) Nucl. Acids Res.18:2188-2193 for tolerance to sulfonylurea herbicides; and the bar genedescribed in DeBlock, et al. (1987) EMBO J. 6:2513-2519 for glufosinateand bialaphos tolerance.

The regeneration, development, and cultivation of plants from varioustransformed explants are well documented in the art. This regenerationand growth process typically includes the steps of selecting transformedcells and culturing those individualized cells through the usual stagesof embryonic development through the rooted plantlet stage. Transgenicembryos and seeds are similarly regenerated. The resulting transgenicrooted shoots are thereafter planted in an appropriate plant growthmedium such as soil. Cells that survive the exposure to the selectiveagent, or cells that have been scored positive in a screening assay, maybe cultured in media that supports regeneration of plants. Developingplantlets are transferred to soil less plant growth mix, and hardenedoff, prior to transfer to a greenhouse or growth chamber for maturation.

The present invention can be used with any transformable cell or tissue.By transformable as used herein is meant a cell or tissue that iscapable of further propagation to give rise to a plant. Those of skillin the art recognize that a number of plant cells or tissues aretransformable in which after insertion of exogenous DNA and appropriateculture conditions the plant cells or tissues can form into adifferentiated plant. Tissue suitable for these purposes can include butis not limited to immature embryos, scutellar tissue, suspension cellcultures, immature inflorescence, shoot meristem, nodal explants, callustissue, hypocotyl tissue, cotyledons, roots, and leaves.

Any suitable plant culture medium can be used. Examples of suitablemedia would include but are not limited to MS-based media (Murashige andSkoog, Physiol. Plant, 15:473-497, 1962) or N6-based media (Chu et al.,Scientia Sinica 18:659, 1975) supplemented with additional plant growthregulators including but not limited to auxins, cytokinins, ABA, andgibberellins. Those of skill in the art are familiar with the variety oftissue culture media, which when supplemented appropriately, supportplant tissue growth and development and are suitable for planttransformation and regeneration. These tissue culture media can eitherbe purchased as a commercial preparation, or custom prepared andmodified. Those of skill in the art are aware that media and mediasupplements such as nutrients and growth regulators for use intransformation and regeneration and other culture conditions such aslight intensity during incubation, pH, and incubation temperatures thatcan be optimized for the particular variety of interest.

One of ordinary skill will appreciate that, after an expression cassetteis stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

Identification of Plants with an Altered Oil Content Phenotype

We used an Arabidopsis activation tagging screen to identify theassociation between the gene we have identified and designated“HIO1002,” (At1g73650, GI#30698954) encoding a protein (GI#18410409),and an altered oil content phenotype (specifically, a high oilphenotype). Splice variants and their corresponding GenBank entries arenoted below. Briefly, and as further described in the Examples, a largenumber of Arabidopsis plants were mutated with the pSKI015 vector, whichcomprises a T-DNA from the Ti plasmid of Agrobacterium tumifaciens, aviral enhancer element, and a selectable marker gene (Weigel et al.,2000). When the T-DNA inserts into the genome of transformed plants, theenhancer element can cause up-regulation genes in the vicinity,generally within about 10 kilobase (kb) of the insertion. T1 plants wereexposed to the selective agent in order to specifically recovertransformed plants that expressed the selectable marker and thereforeharbored T-DNA insertions. Samples of approximately 15-20 T2 seeds werecollected from transformed T1 plants, and lipids were extracted fromwhole seeds. Gas chromatography (GC) analysis was performed to determinefatty acid content and composition of seed samples.

An Arabidopsis line that showed a high-oil phenotype was identified. Theassociation of the HIO1002 gene with the high oil phenotype wasdiscovered by analysis of the genomic DNA sequence flanking the T-DNAinsertion in the identified line. Accordingly, HIO1002 genes and/orpolypeptides may be employed in the development of genetically modifiedplants having a modified oil content phenotype (“a HIO1002 phenotype”).HIO1002 genes may be used in the generation of oilseed crops thatprovide improved oil yield from oilseed processing and in the generationof feed grain crops that provide increased energy for animal feeding.HIO1002 genes may further be used to increase the oil content ofspecialty oil crops, in order to augment yield of desired unusual fattyacids. Transgenic plants that have been genetically modified to expressHIO1002 can be used in the production of oil, wherein the transgenicplants are grown, and oil is obtained from plant parts (e.g. seed) usingstandard methods.

HIO1002 Nucleic Acids and Polypeptides

Arabidopsis HIO1002 nucleic acid sequence is provided in SEQ ID NO: 1and in Genbank entry GI#30698954. The corresponding protein sequence isprovided in SEQ ID NO:2 and in GI#18410409. Two putative splice variantswere also identified which encode proteins that differ from SEQ ID NO:2at the COOH terminus: GI#30698952 (SEQ ID NO:3) with a correspondingprotein sequence as in GI#30698953 (SEQ ID NO:4) that is 100% identicalwith SEQ ID NO:2 up to E288, and ending with LG at amino acid positions289 and 290, respectively; and GI#42572098 (SEQ ID NO:5) with acorresponding protein sequence as in GI#42572099 (SEQ ID NO:6) that is100% identical with SEQ ID NO:2 up to E288, and ending withGRLQNSKEKEVKDD at amino acids 289-302, respectively. Nucleic acidsand/or proteins that are orthologs or paralogs of Arabidopsis HIO11002,are described in Example 3 below.

As used herein, the term “HIO1002 polypeptide” refers to a full-lengthHIO1002 protein or a fragment, derivative (variant), or ortholog thereofthat is “functionally active,” meaning that the protein fragment,derivative, or ortholog exhibits one or more or the functionalactivities associated with the polypeptide of SEQ ID NO:2, 4, or 6. Inone preferred embodiment, a functionally active HIO1002 polypeptidecauses an altered oil content phenotype when mis-expressed in a plant.In a further preferred embodiment, mis-expression of the HIO1002polypeptide causes a high oil phenotype in a plant. In anotherembodiment, a functionally active HIO1002 polypeptide is capable ofrescuing defective (including deficient) endogenous HIO1002 activitywhen expressed in a plant or in plant cells; the rescuing polypeptidemay be from the same or from a different species as that with defectiveactivity. In another embodiment, a functionally active fragment of afull length HIO1002 polypeptide (i.e., a native polypeptide having thesequence of SEQ ID NO:2, 4, or 6, or a naturally occurring orthologthereof) retains one of more of the biological properties associatedwith the full-length HIO1002 polypeptide, such as signaling activity,binding activity, catalytic activity, or cellular or extra-cellularlocalizing activity. A HIO1002 fragment preferably comprises a HIO1002domain, such as a C- or N-terminal or catalytic domain, among others,and preferably comprises at least 10, preferably at least 20, morepreferably at least 25, and most preferably at least 50 contiguous aminoacids of a HIO1002 protein. Functional domains can be identified usingthe PFAM program (Bateman A et al., 1999 Nucleic Acids Res 27:260-262).A preferred HIO1002 fragment comprises of one or more transmembranedomains.

Functionally active variants of full-length HIO1002 polypeptides orfragments thereof include polypeptides with amino acid insertions,deletions, or substitutions that retain one of more of the biologicalproperties associated with the full-length HIO1002 polypeptide. In somecases, variants are generated that change the post-translationalprocessing of a HIO1002 polypeptide. For instance, variants may havealtered protein transport or protein localization characteristics oraltered protein half-life compared to the native polypeptide.

As used herein, the term “HIO1002 nucleic acid” encompasses nucleicacids with the sequence provided in or complementary to the sequenceprovided in SEQ ID NO: 1, 3, or 5, as well as functionally activefragments, derivatives, or orthologs thereof. A HIO1002 nucleic acid ofthis invention may be DNA, derived from genomic DNA or cDNA, or RNA.

In one embodiment, a functionally active HIO1002 nucleic acid encodes oris complementary to a nucleic acid that encodes a functionally activeHIO1002 polypeptide. Included within this definition is genomic DNA thatserves as a template for a primary RNA transcript (i.e., an mRNAprecursor) that requires processing, such as splicing, before encodingthe functionally active HIO1002 polypeptide. A HIO1002 nucleic acid caninclude other non-coding sequences, which may or may not be transcribed;such sequences include 5′ and 3′ UTRs, polyadenylation signals andregulatory sequences that control gene expression, among others, as areknown in the art. Some polypeptides require processing events, such asproteolytic cleavage, covalent modification, etc., in order to becomefully active. Accordingly, functionally active nucleic acids may encodethe mature or the pre-processed HIO1002 polypeptide, or an intermediateform. A HIO1002 polynucleotide can also include heterologous codingsequences, for example, sequences that encode a marker included tofacilitate the purification of the fused polypeptide, or atransformation marker.

In another embodiment, a functionally active HIO1002 nucleic acid iscapable of being used in the generation of loss-of-function HIO1002phenotypes, for instance, via antisense suppression, co-suppression,etc.

In one preferred embodiment, a HIO1002 nucleic acid used in the methodsof this invention comprises a nucleic acid sequence that encodes or iscomplementary to a sequence that encodes a HIO1002 polypeptide having atleast 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identityto the polypeptide sequence presented in SEQ ID NO:2, 4 or 6.

In another embodiment a HIO1002 polypeptide of the invention comprises apolypeptide sequence with at least 50% or 60% identity to the HIO1002polypeptide sequence of SEQ ID NO:2, 4 or 6, and may have at least 70%,80%, 85%, 90% or 95% or more sequence identity to the HIO1002polypeptide sequence of SEQ ID NO:2, 4, or 6, such as one or moretransmembrane domains. In another embodiment, a HIO1002 polypeptidecomprises a polypeptide sequence with at least 50%, 60%, 70%, 80%, 85%,90% or 95% or more sequence identity to a functionally active fragmentof the polypeptide presented in SEQ ID NO:2, 4, or 6. In yet anotherembodiment, a HIO1002 polypeptide comprises a polypeptide sequence withat least 50%, 60%, 70%, 80%, or 90% identity to the polypeptide sequenceof SEQ ID NO:2, 4 or 6 over its entire length and comprises of one ormore transmembrane domains.

In another aspect, a HIO1002 polynucleotide sequence is at least 50% to60% identical over its entire length to the HIO1002 nucleic acidsequence presented as SEQ ID NO: 1, 3, or 5, or nucleic acid sequencesthat are complementary to such a HIO1002 sequence, and may comprise atleast 70%, 80%, 85%, 90% or 95% or more sequence identity to the HIO1002sequence presented as SEQ ID NO: 1, 3, or 5, or a functionally activefragment thereof, or complementary sequences.

As used herein, “percent (%) sequence identity” with respect to aspecified subject sequence, or a specified portion thereof, is definedas the percentage of nucleotides or amino acids in the candidatederivative sequence identical with the nucleotides or amino acids in thesubject sequence (or specified portion thereof), after aligning thesequences and introducing gaps, if necessary to achieve the maximumpercent sequence identity, as generated by the program WU-BLAST-2.0a19(Altschul et al., J. Mol. Biol. (1997) 215:403-410) with searchparameters set to default values. The HSP S and HSP S2 parameters aredynamic values and are established by the program itself depending uponthe composition of the particular sequence and composition of theparticular database against which the sequence of interest is beingsearched. A “% identity value” is determined by the number of matchingidentical nucleotides or amino acids divided by the sequence length forwhich the percent identity is being reported. “Percent (%) amino acidsequence similarity” is determined by doing the same calculation as fordetermining % amino acid sequence identity, but including conservativeamino acid substitutions in addition to identical amino acids in thecomputation. A conservative amino acid substitution is one in which anamino acid is substituted for another amino acid having similarproperties such that the folding or activity of the protein is notsignificantly affected. Aromatic amino acids that can be substituted foreach other are phenylalanine, tryptophan, and tyrosine; interchangeablehydrophobic amino acids are leucine, isoleucine, methionine, and valine;interchangeable polar amino acids are glutamine and asparagine;interchangeable basic amino acids are arginine, lysine and histidine;interchangeable acidic amino acids are aspartic acid and glutamic acid;and interchangeable small amino acids are alanine, serine, threonine,cysteine and glycine.

Derivative nucleic acid molecules of the subject nucleic acid moleculesinclude sequences that selectively hybridize to the nucleic acidsequence of SEQ ID NO: 1. The stringency of hybridization can becontrolled by temperature, ionic strength, pH, and the presence ofdenaturing agents such as formamide during hybridization and washing.Conditions routinely used are well known (see, e.g., Current Protocol inMolecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons, Publishers(1994); Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989)).In some embodiments, a nucleic acid molecule of the invention is capableof hybridizing to a nucleic acid molecule containing the nucleotidesequence of SEQ ID NO: 1 under stringent hybridization conditions thatare: prehybridization of filters containing nucleic acid for 8 hours toovernight at 65° C. in a solution comprising 6× single strength citrate(SSC) (1×SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5× Denhardt'ssolution, 0.05% sodium pyrophosphate and 100 μg/ml herring sperm DNA;hybridization for 18-20 hours at 65° C. in a solution containing 6×SSC,1× Denhardt's solution, 100 μg/ml yeast tRNA and 0.05% sodiumpyrophosphate; and washing of filters at 65° C. for 1 h in a solutioncontaining 0.1×SSC and 0.1% SDS (sodium dodecyl sulfate). In otherembodiments, moderately stringent hybridization conditions are used thatare: pretreatment of filters containing nucleic acid for 6 h at 40° C.in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCI (pH 7.5),5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmonsperm DNA; hybridization for 18-20 h at 40° C. in a solution containing35% formamide, 5×SSC, 50 mM Tris-HCI (pH 7.5), 5 mM EDTA, 0.02% PVP,0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, and 10% (wt/vol)dextran sulfate; followed by washing twice for 1 hour at 55° C. in asolution containing 2×SSC and 0.1% SDS. Alternatively, low stringencyconditions can be used that comprise: incubation for 8 hours toovernight at 37° C. in a solution comprising 20% formamide, 5×SSC, 50 mMsodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate,and 20 μg/ml denatured sheared salmon sperm DNA; hybridization in thesame buffer for 18 to 20 hours; and washing of filters in 1×SSC at about37° C. for 1 hour.

As a result of the degeneracy of the genetic code, a number ofpolynucleotide sequences encoding a HIO1002 polypeptide can be produced.For example, codons may be selected to increase the rate at whichexpression of the polypeptide occurs in a particular host species, inaccordance with the optimum codon usage dictated by the particular hostorganism (see, e.g., Nakamura et al, 1999). Such sequence variants maybe used in the methods of this invention.

The methods of the invention may use orthologs of the ArabidopsisHIO1002. Methods of identifying the orthologs in other plant species areknown in the art. Normally, orthologs in different species retain thesame function, due to presence of one or more protein motifs and/or3-dimensional structures. In evolution, when a gene duplication eventfollows speciation, a single gene in one species, such as Arabidopsis,may correspond to multiple genes (paralogs) in another. As used herein,the term “orthologs” encompasses paralogs. When sequence data isavailable for a particular plant species, orthologs are generallyidentified by sequence homology analysis, such as BLAST analysis,usually using protein bait sequences. Sequences are assigned as apotential ortholog if the best hit sequence from the forward BLASTresult retrieves the original query sequence in the reverse BLAST(Huynen M A and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen MA et al., Genome Research (2000) 10:1204-1210).

Programs for multiple sequence alignment, such as CLUSTAL (Thompson J Det al, 1994, Nucleic Acids Res 22:4673-4680) may be used to highlightconserved regions and/or residues of orthologous proteins and togenerate phylogenetic trees. In a phylogenetic tree representingmultiple homologous sequences from diverse species (e.g., retrievedthrough BLAST analysis), orthologous sequences from two speciesgenerally appear closest on the tree with respect to all other sequencesfrom these two species. Structural threading or other analysis ofprotein folding (e.g., using software by ProCeryon, Biosciences,Salzburg, Austria) may also identify potential orthologs. Nucleic acidhybridization methods may also be used to find orthologous genes and arepreferred when sequence data are not available. Degenerate PCR andscreening of cDNA or genomic DNA libraries are common methods forfinding related gene sequences and are well known in the art (see, e.g.,Sambrook, 1989; Dieffenbach and Dveksler, 1995). For instance, methodsfor generating a cDNA library from the plant species of interest andprobing the library with partially homologous gene probes are describedin Sambrook et al. A highly conserved portion of the Arabidopsis HIO1002coding sequence may be used as a probe. HIO1002 ortholog nucleic acidsmay hybridize to the nucleic acid of SEQ ID NO: 1 under high, moderate,or low stringency conditions. After amplification or isolation of asegment of a putative ortholog, that segment may be cloned and sequencedby standard techniques and utilized as a probe to isolate a completecDNA or genomic clone. Alternatively, it is possible to initiate an ESTproject to generate a database of sequence information for the plantspecies of interest. In another approach, antibodies that specificallybind known HIO1002 polypeptides are used for ortholog isolation (see,e.g., Harlow and Lane, 1988, 1999). Western blot analysis can determinethat a HIO1002 ortholog (i.e., an orthologous protein) is present in acrude extract of a particular plant species. When reactivity isobserved, the sequence encoding the candidate ortholog may be isolatedby screening expression libraries representing the particular plantspecies. Expression libraries can be constructed in a variety ofcommercially available vectors, including lambda gt11, as described inSambrook, et al., 1989. Once the candidate ortholog(s) are identified byany of these means, candidate orthologous sequence are used as bait (the“query”) for the reverse BLAST against sequences from Arabidopsis orother species in which HIO1002 nucleic acid and/or polypeptide sequenceshave been identified.

HIO1002 nucleic acids and polypeptides may be obtained using anyavailable method. For instance, techniques for isolating cDNA or genomicDNA sequences of interest by screening DNA libraries or by usingpolymerase chain reaction (PCR), as previously described, are well knownin the art. Alternatively, nucleic acid sequence may be synthesized. Anyknown method, such as site directed mutagenesis (Kunkel T A et al.,1991), may be used to introduce desired changes into a cloned nucleicacid.

In general, the methods of the invention involve incorporating thedesired form of the HIO1002 nucleic acid into a plant expression vectorfor transformation of in plant cells, and the HIO1002 polypeptide isexpressed in the host plant.

An isolated HIO1002 nucleic acid molecule is other than in the form orsetting in which it is found in nature and is identified and separatedfrom least one contaminant nucleic acid molecule with which it isordinarily associated in the natural source of the HIO1002 nucleic acid.However, an isolated HIO1002 nucleic acid molecule includes HIO1002nucleic acid molecules contained in cells that ordinarily expressHIO1002 where, for example, the nucleic acid molecule is in achromosomal location different from that of natural cells.

Generation of Genetically Modified Plants with an Altered Oil ContentPhenotype

HIO1002 nucleic acids and polypeptides may be used in the generation ofgenetically modified plants having a modified oil content phenotype. Asused herein, a “modified oil content phenotype” may refer to modifiedoil content in any part of the plant; the modified oil content is oftenobserved in seeds. In a preferred embodiment, altered expression of theHIO1002 gene in a plant is used to generate plants with a high oilphenotype.

The methods described herein are generally applicable to all plants.Although activation tagging and gene identification is carried out inArabidopsis, the HIO1002 gene (or an ortholog, variant or fragmentthereof) may be expressed in any type of plant. In a preferredembodiment, the invention is directed to oil-producing plants, whichproduce and store triacylglycerol in specific organs, primarily inseeds. Such species include soybean (Glycine max), rapeseed and canola(including Brassica napus, B. campestris), sunflower (Helianthus annus),cotton (Gossypium hirsutum), corn (Zea mays), cocoa (Theobroma cacao),safflower (Carthamus tinctorius), oil palm (Elaeis guineensis), coconutpalm (Cocos nucifera), flax (Linum usitatissimum), castor (Ricinuscommunis) and peanut (Arachis hypogaea). The invention may also bedirected to fruit- and vegetable-bearing plants, grain-producing plants,nut-producing plants, rapid cycling Brassica species, alfalfa (Medicagosativa), tobacco (Nicotiana), turfgrass (Poaceae family), other foragecrops, and wild species that may be a source of unique fatty acids.

The skilled artisan will recognize that a wide variety of transformationtechniques exist in the art, and new techniques are continually becomingavailable. Any technique that is suitable for the target host plant canbe employed within the scope of the present invention. For example, theconstructs can be introduced in a variety of forms including, but notlimited to as a strand of DNA, in a plasmid, or in an artificialchromosome. The introduction of the constructs into the target plantcells can be accomplished by a variety of techniques, including, but notlimited to Agrobacterium-mediated transformation, electroporation,microinjection, microprojectile bombardment calcium-phosphate-DNAco-precipitation or liposome-mediated transformation of a heterologousnucleic acid. The transformation of the plant is preferably permanent,i.e. by integration of the introduced expression constructs into thehost plant genome, so that the introduced constructs are passed ontosuccessive plant generations. Depending upon the intended use, aheterologous nucleic acid construct comprising an HIO1002 polynucleotidemay encode the entire protein or a biologically active portion thereof.

In one embodiment, binary Ti-based vector systems may be used totransfer polynucleotides. Standard Agrobacterium binary vectors areknown to those of skill in the art, and many are commercially available(e.g., pBI121 Clontech Laboratories, Palo Alto, Calif.). A construct orvector may include a plant promoter to express the nucleic acid moleculeof choice. In a preferred embodiment, the promoter is a plant promoter.

The optimal procedure for transformation of plants with Agrobacteriumvectors will vary with the type of plant being transformed. Exemplarymethods for Agrobacterium-mediated transformation include transformationof explants of hypocotyl, shoot tip, stem or leaf tissue, derived fromsterile seedlings and/or plantlets. Such transformed plants may bereproduced sexually, or by cell or tissue culture. Agrobacteriumtransformation has been previously described for a large number ofdifferent types of plants and methods for such transformation may befound in the scientific literature. Of particular relevance are methodsto transform commercially important crops, such as rapeseed (De Block etal., 1989), sunflower (Everett et al., 1987), and soybean (Christou etal., 1989; Kline et al., 1987).

Expression (including transcription and translation) of HIO1002 may beregulated with respect to the level of expression, the tissue type(s)where expression takes place and/or developmental stage of expression. Anumber of heterologous regulatory sequences (e.g., promoters andenhancers) are available for controlling the expression of a HIO1002nucleic acid. These include constitutive, inducible and regulatablepromoters, as well as promoters and enhancers that control expression ina tissue- or temporal-specific manner. Exemplary constitutive promotersinclude the raspberry E4 promoter (U.S. Pat. Nos. 5,783,393 and5,783,394), the nopaline synthase (NOS) promoter (Ebert et al., Proc.Natl. Acad. Sci. (U.S.A.) 84:5745-5749, 1987), the octopine synthase(OCS) promoter (which is carried on tumor-inducing plasmids ofAgrobacterium tumefaciens), the caulimovirus promoters such as thecauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., Plant Mol.Biol. 9:315-324, 1987) and the CaMV 35S promoter (Odell et al., Nature313:810-812, 1985 and Jones J D et al, 1992), the melon actin promoter(published PCT application WO0056863), the figwort mosaic virus35S-promoter (U.S. Pat. No. 5,378,619), the light-inducible promoterfrom the small subunit of ribulose-1,5-bis-phosphate carboxylase(ssRUBISCO), the Adh promoter (Walker et al., Proc. Natl. Acad. Sci.(U.S.A.) 84:6624-6628, 1987), the sucrose synthase promoter (Yang etal., Proc. Natl Acad. Sci. (U.S.A.) 87:4144-4148, 1990), the R genecomplex promoter (Chandler et al., The Plant Cell 1:1175-1183, 1989),the chlorophyll a/b binding protein gene promoter the CsVMV promoter(Verdaguer B et al., 1998); these promoters have been used to create DNAconstructs that have been expressed in plants, e.g., PCT publication WO84/02913. Exemplary tissue-specific promoters include the tomato E4 andE8 promoters (U.S. Pat. No. 5,859,330) and the tomato 2AII gene promoter(Van Haaren M J J et al., 1993).

In one preferred embodiment, HIO1002 expression is under control ofregulatory sequences from genes whose expression is associated withearly seed and/or embryo development. Indeed, in a preferred embodiment,the promoter used is a seed-enhanced promoter. Examples of suchpromoters include the 5′ regulatory regions from such genes as napin(Kridl et al., Seed Sci. Res. 1:209:219, 1991), globulin (Belanger andKriz, Genet., 129: 863-872, 1991, GenBank Accession No. L22295), gammazein Z 27 (Lopes et al., Mol Gen Genet., 247:603-613, 1995), L3 oleosinpromoter (U.S. Pat. No. 6,433,252), phaseolin (Bustos et al., PlantCell, 1(9):839-853, 1989), arcelin5 (US 2003/0046727), a soybean 7Spromoter, a 7Sα promoter (US 2003/0093828), the soybean 7Sα′ betaconglycinin promoter, a 7S α′ promoter (Beachy et al., EMBO J, 4:3047,1985; Schuler et al., Nucleic Acid Res., 10(24):8225-8244, 1982),soybean trypsin inhibitor (Riggs et al., Plant Cell 1(6):609-621, 1989),ACP (Baerson et al., Plant Mol. Biol., 22(2):255-267, 1993),stearoyl-ACP desaturase (Slocombe et al., Plant Physiol. 104(4):167-176,1994), soybean a′ subunit of β-conglycinin (Chen et al., Proc. Natl.Acad. Sci. 83:8560-8564, 1986), Vicia faba USP (β-Vf.Usp, SEQ ID NO: 1,2, and 3 in (US 2003/229918) and Zea mays L3 oleosin promoter (Hong etal., Plant Mol. Biol., 34(3):549-555, 1997). Also included are thezeins, which are a group of storage proteins found in corn endosperm.Genomic clones for zein genes have been isolated (Pedersen et al., Cell29:1015-1026, 1982; and Russell et al., Transgenic Res. 6(2):157-168)and the promoters from these clones, including the 15 kD, 16 kD, 19 kD,22 kD, 27 kD and genes, could also be used. Other promoters known tofunction, for example, in corn include the promoters for the followinggenes: waxy, Brittle, Shrunken 2, Branching enzymes I and II, starchsynthases, debranching enzymes, oleosins, glutelins and sucrosesynthases. Legume genes whose promoters are associated with early seedand embryo development include V. faba legumin (Baumlein et al., 1991,Mol Gen Genet 225:121-8; Baumlein et al., 1992, Plant J 2:233-9), V.faba usp (Fiedler et al., 1993, Plant Mol Biol 22:669-79), peaconvicilin (Bown et al., 1988, Biochem J 251:717-26), pea lectin(dePater et al., 1993, Plant Cell 5:877-86), P. vulgaris beta phaseolin(Bustos et al., 1991, EMBO J 10: 1469-79), P. vulgaris DLEC2 and PHS[beta] (Bobb et al, 1997, Nucleic Acids Res 25:641-7), and soybeanbeta-Conglycinin, 7S storage protein (Chamberland et al., 1992, PlantMol Biol 19:93749).

Cereal genes whose promoters are associated with early seed and embryodevelopment include rice glutelin (“GluA-3,” Yoshihara and Takaiwa,1996, Plant Cell Physiol 37:107-11; “GluB-1,” Takaiwa et al., 1996,Plant Mol Biol 30:1207-21; Washida et al., 1999, Plant Mol Biol 40:1-12;“Gt3,” Leisy et al., 1990, Plant Mol Biol 14:41-50), rice prolamin (Zhou& Fan, 1993, Transgenic Res 2:141-6), wheat prolamin (Hammond-Kosack etal., 1993, EMBO J 12:545-54), maize zein (Z4, Matzke et al., 1990, PlantMol Biol 14:323-32), and barley B-hordeins (Entwistle et al., 1991,Plant Mol Biol 17:1217-31).

Other genes whose promoters are associated with early seed and embryodevelopment include oil palm GLO7A (7S globulin, Morcillo et al., 2001,Physiol Plant 112:233-243), Brassica napus napin, 2S storage protein,and napA gene (Josefsson et al., 1987, J Biol Chem 262:12196-201;Stalberg et al., 1993, Plant Mol Biol 1993 23:671-83; Ellerstrom et al.,1996, Plant Mol Biol 32:1019-27), Brassica napus oleosin (Keddie et al.,1994, Plant Mol Biol 24:327-40), Arabidopsis oleosin (Plant et al.,1994, Plant Mol Biol 25:193-205), Arabidopsis FAE1 (Rossak et al., 2001,Plant Mol Biol 46:717-25), Canavalia gladiata conA (Yamamoto et al.,1995, Plant Mol Biol 27:729-41), and Catharanthus roseus strictosidinesynthase (Str, Ouwerkerk and Memelink, 1999, Mol Gen Genet 261:635-43).In another preferred embodiment, regulatory sequences from genesexpressed during oil biosynthesis are used (see, e.g., U.S. Pat. No.5,952,544). Alternative promoters are from plant storage protein genes(Bevan et al, 1993, Philos Trans R Soc Lond B Biol Sci 342:209-15).Additional promoters that may be utilized are described, for example, inU.S. Pat. Nos. 5,378,619; 5,391,725; 5,428,147; 5,447,858; 5,608,144;5,608,144; 5,614,399; 5,633,441; 5,633,435; and 4,633,436.

In yet another aspect, in some cases it may be desirable to inhibit theexpression of endogenous HIO1002 in a host cell. Exemplary methods forpracticing this aspect of the invention include, but are not limited toantisense suppression (Smith, et al., 1988; van der Krol et al., 1988);co-suppression (Napoli, et al., 1990); ribozymes (PCT Publication WO97/10328); and combinations of sense and antisense (Waterhouse, et al.,1998). Methods for the suppression of endogenous sequences in a hostcell typically employ the transcription or transcription and translationof at least a portion of the sequence to be suppressed. Such sequencesmay be homologous to coding as well as non-coding regions of theendogenous sequence. Antisense inhibition may use the entire cDNAsequence (Sheehy et al., 1988), a partial cDNA sequence includingfragments of 5′ coding sequence, (Cannon et al., 1990), or 3′ non-codingsequences (Ch'ng et al., 1989). Cosuppression techniques may use theentire cDNA sequence (Napoli et al., 1990; van der Krol et al., 1990),or a partial cDNA sequence (Smith et al., 1990).

Standard molecular and genetic tests may be performed to further analyzethe association between a gene and an observed phenotype. Exemplarytechniques are described below.

1. DNA/RNA Analysis

The stage- and tissue-specific gene expression patterns in mutant versuswild-type lines may be determined, for instance, by in situhybridization. Analysis of the methylation status of the gene,especially flanking regulatory regions, may be performed. Other suitabletechniques include overexpression, ectopic expression, expression inother plant species and gene knock-out (reverse genetics, targetedknock-out, viral induced gene silencing [VIGS, see Baulcombe D, 1999]).

In a preferred application expression profiling, generally by microarrayanalysis, is used to simultaneously measure differences or inducedchanges in the expression of many different genes. Techniques formicroarray analysis are well known in the art (Schena M et al., Science(1995) 270:467-470; Baldwin D et al., 1999; Dangond F, Physiol Genomics(2000) 2:53-58; van Hal N L et al., J Biotechnol (2000) 78:271-280;Richmond T and Somerville S, Curr Opin Plant Biol (2000) 3:108-116).Expression profiling of individual tagged lines may be performed. Suchanalysis can identify other genes that are coordinately regulated as aconsequence of the overexpression of the gene of interest, which mayhelp to place an unknown gene in a particular pathway.

2. Gene Product Analysis

Analysis of gene products may include recombinant protein expression,antisera production, immunolocalization, biochemical assays forcatalytic or other activity, analysis of phosphorylation status, andanalysis of interaction with other proteins via yeast two-hybrid assays.

3. Pathway Analysis

Pathway analysis may include placing a gene or gene product within aparticular biochemical, metabolic or signaling pathway based on itsmis-expression phenotype or by sequence homology with related genes.Alternatively, analysis may comprise genetic crosses with wild-typelines and other mutant lines (creating double mutants) to order the genein a pathway, or determining the effect of a mutation on expression ofdownstream “reporter” genes in a pathway.

Generation of Mutated Plants with an Altered Oil Content Phenotype

The invention further provides a method of identifying plants that havemutations in endogenous HIO1002 that confer altered oil content, andgenerating altered oil content progeny of these plants that are notgenetically modified. In one method, called “TILLING” (for targetinginduced local lesions in genomes), mutations are induced in the seed ofa plant of interest, for example, using EMS treatment. The resultingplants are grown and self-fertilized, and the progeny are used toprepare DNA samples. HIO1002-specific PCR is used to identify whether amutated plant has a HIO1002 mutation. Plants having HIO1002 mutationsmay then be tested for altered oil content, or alternatively, plants maybe tested for altered oil content, and then HIO1002-specific PCR is usedto determine whether a plant having altered oil content has a mutatedHIO1002 gene. TILLING can identify mutations that may alter theexpression of specific genes or the activity of proteins encoded bythese genes (see Colbert et al (2001) Plant Physiol 126:480-484;McCallum et al (2000) Nature Biotechnology 18:455-457).

In another method, a candidate gene/Quantitative Trait Locus (QTLs)approach can be used in a marker-assisted breeding program to identifyalleles of or mutations in the HIO1002 gene or orthologs of HIO1002 thatmay confer altered oil content (see Bert et al., Theor Appl Genet. 2003June; 107(1):181-9; and Lionneton et al, Genome. 2002 December;45(6):1203-15). Thus, in a further aspect of the invention, a HIO1002nucleic acid is used to identify whether a plant having altered oilcontent has a mutation in endogenous HIO1002 or has a particular allelethat causes altered oil content.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention. All publications cited herein are expressly incorporatedherein by reference for the purpose of describing and disclosingcompositions and methodologies that might be used in connection with theinvention. All cited patents, patent applications, and sequenceinformation in referenced websites and public databases are alsoincorporated by reference.

EXAMPLES Example 1

Generation of Plants with a HIO1002 Phenotype by Transformation with anActivation Tagging Construct

Mutants were generated using the activation tagging “ACTTAG” vector,pSKI015 (GI#6537289; Weigel D et al., 2000). Standard methods were usedfor the generation of Arabidopsis transgenic plants, and wereessentially as described in published application PCT WO0183697.Briefly, T0 Arabidopsis (Col-0) plants were transformed withAgrobacterium carrying the pSKI015 vector, which comprises T-DNA derivedfrom the Agrobacterium Ti plasmid, an herbicide resistance selectablemarker gene, and the 4×CaMV 35S enhancer element. Transgenic plants wereselected at the T1 generation based on herbicide resistance.

T3 seed was analyzed by Near Infrared Spectroscopy (NIR) at the time ofharvest. NIR infrared spectra were captured using a Bruker 22 N/F.Bruker Software was used to estimate total seed oil and total seedprotein content using data from NIR analysis and reference methodsaccording to the manufacturers instructions. Oil contents predicted byour calibration (ren oil 1473 1d+sline.q2, Predicts Hexane ExtractedOil), which followed the general method of AOCS Procedure AM1-92,Official Methods and Recommended Practices of the American Oil ChemistsSociety, 5th Ed., AOCS, Champaign, Ill., were compared for 38,090individual ACTTAG lines. Subsequent to seed compositional analysis, theposition of the ACTTAG element in the genome of in each line wasdetermined by inverse PCR and sequencing. 38,090 lines with recoveredflanking sequences were considered in this analysis.

Since the 38,090 lines were planted and grown over a 12-month period,the seed oil content values were normalized to minimize the effect ofenvironmental differences which may alter seed oil content. The averageseed oil content and its standard deviation, for each day lines wereplanted, were calculated. The seed oil content was expressed as a“relative standard deviation distance” (SD distance) which wascalculated by subtracting the average seed oil content for the plantingday from seed oil content for each line and dividing the difference bythe standard deviation for that day. This normalization allowscomparison of seed oil content in seed from plants grown throughout theyear.

Genes that cause a high seed oil phenotype when over-expressed wereidentified by evaluating all of the genes affected by ACTTAG elements inthe 38,090 lines. This was accomplished by the following procedure;first, the genes likely to be activated by the ACTTAG element in eachline were identified and the seed oil content of the line was assignedto these genes; second, the seed oil content when a particular gene isover-expressed was determined by averaging the individual seed oilvalues for each gene. Since 38,090 lines were evaluated and each elementaffects an average of 2.5 genes, each gene will have an average of 4seed oil values. The genes with the highest average SD distance weredetermined to be those that cause a high seed oil phenotype whenover-expressed.

Seed from plants over-expressing At1g73650 have an oil content of 128%of the planting day average, as is shown in the following Table 1. TABLE1 n (# of relative ACTTAG lines Standard deviation standard planted forthe Average oil of the oil deviation plant Planting date with NIR SeedOil content of the content for the Tair distance count Description LineID date measurement) content (%) planting date (%) planting dateAt1g73650 2.960951 1 expressed W000203274 Oct. 7, 2002 1184 32.01625.025 2.361 protein

Example 2

Characterization of the T-DNA Insertion in Plants Exhibiting the AlteredOil Content Phenotype.

We performed standard molecular analyses, essentially as described inpatent application PCT WO0183697, to determine the site of the T-DNAinsertion associated with the altered oil content phenotype. Briefly,genomic DNA was extracted from plants exhibiting the altered oil contentphenotype. PCR, using primers specific to the pSKI015 vector, confirmedthe presence of the 35S enhancer in plants from the HIO1002 oil line,and Southern blot analysis verified the genomic integration of theACTTAG T-DNA and showed the presence of the T-DNA insertions in each ofthe transgenic lines.

Inverse PCR was used to recover genomic DNA flanking the T-DNAinsertion, which was then subjected to sequence analysis using a basicBLASTN search and/or a search of the Arabidopsis Information Resource(TAIR) database (available at the arabidopsis.org website).

Example 3

Recapitulation of HIO1002 Phenotype

To test whether over-expression of At1g73650 causes a high seed oilphenotype, oil content in seeds from transgenic plants over-expressingthis gene was compared with oil content in seeds from non-transgeniccontrol plants. To do this, At1g73650 was cloned into a planttransformation vector behind the seed specific CsVMV promoter andtransformed into Arabidopsis plants using the floral dip method. Theplant transformation vector contains the nptII gene driven by the RE4promoter, to provide resistance to kanamyacin, and serve as a selectablemarker. Seed from the transformed plants were plated on agar mediumcontaining kanamycin. After 7 days, transgenic plants were identified ashealthy green plants and transplanted to soil. Non-transgenic controlplants were germinated on agar medium, allowed to grow for 7 days andthen transplanted to soil. Twenty-two transgenic seedlings and 10non-transgenic control plants were transplanted to random positions inthe same 32 cell flat. The plants were grown to maturity, allowed toself-fertilize and set seed. Seed was harvested from each plant and itsoil content estimated by Near Infrared (NIR) Spectroscopy using methodspreviously described. The percent oil in the seed harvested from eachplant as determined by NIR spectroscopy is presented in Table 3. TheRelative Oil value is determined by dividing the predicted oil value bythe average oil value in control seed (i.e. seed from plants without thetrangene).

The effect of over-expression of At1g73650 on seed oil has been testedin two experiments. In both experiments, the plants over-expressingAt1g73650 had higher seed oil content than the control plants grown inthe same flat. Across the experiments, the average seed oil content ofplants over-expressing At1g73650 was 4.5% greater than the untransformedcontrols. The seed oil content in plants over-expressing At1g73650 wassignificantly greater than non-transgenic control plants (two-way ANOVA;P=0.0160). TABLE 2 Percent Relative Experiment Plant Transgene Oil Oil 1DX07122001 CsVMV:At1g73650 29.89 110.48 1 DX07122002 CsVMV:At1g7365026.7 98.68 1 DX07122003 CsVMV:At1g73650 27.33 101.03 1 DX07122004CsVMV:At1g73650 26.13 96.59 1 DX07122005 CsVMV:At1g73650 24.09 89.04 1DX07122006 CsVMV:At1g73650 27.38 101.2 1 DX07122007 CsVMV:At1g7365030.58 113.05 1 DX07122009 CsVMV:At1g73650 28.05 103.68 1 DX07122010CsVMV:At1g73650 28.15 104.06 1 DX07122011 CsVMV:At1g73650 26 96.11 1DX07122012 CsVMV:At1g73650 25.16 93 1 DX07122013 CsVMV:At1g73650 25.6494.78 1 DX07122014 CsVMV:At1g73650 27.04 99.97 1 DX07122015CsVMV:At1g73650 25.86 95.6 1 DX07122016 CsVMV:At1g73650 26.26 97.08 1DX07122017 CsVMV:At1g73650 30.09 111.23 1 DX07122018 CsVMV:At1g7365028.19 104.22 1 DX07122019 CsVMV:At1g73650 29.95 110.7 1 DX07122020CsVMV:At1g73650 29 107.18 1 DX07122021 CsVMV:At1g73650 28.88 106.77 1DX07122022 CsVMV:At1g73650 27.29 100.88 1 DX07140001 None 26.33 97.33 1DX07140002 None 26.54 98.12 1 DX07140003 None 28.19 104.21 1 DX07140004None 26.17 96.72 1 DX07140005 None 26.05 96.29 1 DX07140006 None 25.3993.86 1 DX07140007 None 25.87 95.64 1 DX07140008 None 31.93 118.04 1DX07140009 None 28.13 103.99 1 DX07140010 None 25.92 95.79 2 DX07167001CsVMV:At1g73650 31.77 106.66 2 DX07167002 CsVMV:At1g73650 32.21 108.14 2DX07167003 CsVMV:At1g73650 31.63 106.18 2 DX07167004 CsVMV:At1g7365030.2 101.37 2 DX07167005 CsVMV:At1g73650 30.5 102.4 2 DX07167006CsVMV:At1g73650 31.62 106.17 2 DX07167007 CsVMV:At1g73650 30.55 102.57 2DX07167008 CsVMV:At1g73650 36.57 122.77 2 DX07167009 CsVMV:At1g7365032.7 109.78 2 DX07167010 CsVMV:At1g73650 29.27 98.25 2 DX07167011CsVMV:At1g73650 30 100.71 2 DX07167012 CsVMV:At1g73650 29.58 99.29 2DX07167013 CsVMV:At1g73650 33.75 113.31 2 DX07167014 CsVMV:At1g7365030.63 102.84 2 DX07167015 CsVMV:At1g73650 35.07 117.74 2 DX07167016CsVMV:At1g73650 34.49 115.79 2 DX07167017 CsVMV:At1g73650 32.99 110.75 2DX07167018 CsVMV:At1g73650 33.06 110.99 2 DX07167019 CsVMV:At1g7365031.52 105.8 2 DX07167020 CsVMV:At1g73650 32.8 110.13 2 DX07167022CsVMV:At1g73650 30.37 101.94 2 DX07185001 None 29.9 100.39 2 DX07185002None 28.47 95.59 2 DX07185003 None 28.83 96.79 2 DX07185004 None 29.0397.45 2 DX07185005 None 29.05 97.51 2 DX07185007 None 27.27 91.56 2DX07185008 None 31.06 104.26 2 DX07185009 None 32.22 108.18 2 DX07185010None 32.25 108.28

Example 4

Analysis of Arabidopsis HIO1002 Sequence

Sequence analyses were performed with BLAST (Altschul et al., 1990, J.Mol. Biol. 215:403-410), PFAM (Bateman et al., 1999, Nucleic Acids Res27:260-262), PSORT (Nakai K, and Horton P, 1999, Trends Biochem Sci24:34-6), and/or CLUSTAL (Thompson J D et al., 1994, Nucleic Acids Res22:4673-4680).

TBLASTN Against ESTs:

The candidate gene At1g73650 is supported by the full-length cDNAsGI:15809973 and GI:27363285. There are many ESTs from diverse plantspecies showing similarity to At1g73650. Where possible, ESTs contigs ofeach species were made. The top hit for each of the following speciesare listed below and included in the orthologue Table 2: Triticumaestivum, Glycine max, Mentha×piperita, Populus tremula, Oryza sativa,Lycopersicon esculentum, Solanum tuberosum, and Beta vulgaris.

1. Sugar Beet ESTs with the Following GenBank IDS:

gi|26121630

2. Potato ESTs with the Following GenBank IDS:

gi|12588047

gi|15259347

3. Tomato ESTs with the Following GenBank IDS:

gi|5896833

gi|5896833

gi|5896833

gi|12628275

gi|12628275

gi|12628275

gi|16242572

4. Rice ESTs with the Following GenBank IDS:

gi|5456487

gi|13420908

gi|700187

5. Poplar ESTs with the Following GenBank IDS:

gi|3857688

gi|23959842

gi|23960044

gi|23963690

gi|23997967

gi|23998879

gi|24062476

gi|24065210

gi|28609230

6. Soybean ESTs with the Following GenBank IDS:

gi|9205433

gi|12488109

gi|12773915

gi|15286042

gi|18731515

7. Corn ESTs with the Following GenBank IDS:

gi|5739680

gi|21208932

gi|407242

gi|407243

8. Cotton ESTs with the Following GenBank IDS:

gi|3326250

9. Wheat ESTs with the Following GenBank IDS:

gi|9742370

gi|20309688

gi|25194283

gi|25235692

gi|25237006

gi|25243036

gi|25261519

gi|25280706

gi|25431065

gi|25547029

gi|25560875

gi|32546080

gi|32670437

BLASTP Against Amino Acids:

The protein At1g73650 has homology to proteins from other organisms. Thetop seven BLAST results for At1g73650 are listed below and are includedin the Orthologue Table 2 below.

1. One EST contig from Beta vulgaris

-   -   ex |13978852|exs|13978851

2. One EST contig from Solanum tubersom

-   -   ex|11208765|exs|11208764

3. One EST contig from Lycopersicon esculentum

-   -   ex|5891894|exs|5891893

4. One EST contig from Oryza sativa

-   -   ex|7258057|exs|7258056

5. One EST contig from Populus tremula

-   -   ex|10187045|exs|10187044

6. One EST contig from Glycine max

-   -   ex|9156811|exs|9156810

7. One EST contig from Zea mays

-   -   ex|7766057|exs|7766056

8. One EST contig from Gossypium hirsutum

-   -   ex|9283238|exs|9283237

9. One EST contig from Triticum aestivum

ex|11024927|exs|1024926 TABLE 3 Ortholog Gene % ID to Score(s) (BLAST,Clustal, Name Species GI # HIO1002 etc.) One EST Solarium gi|12588047Length: 585 TBLASTN contig tubersom gi|15259347 Identities: 0.653 Score:691 from consensus: SEQ Positives: 0.774 Probability: 2.300000e−68potato ID NO: 7 Frames: 3 One EST Beta gi|26121630 Length: 504 TBLASTNcontig vulgaris Identities: 0.667 Score: 577 from Positives: 0.758Probability: 1.300000e−56 sugar Frames: 1 beet One EST Glycine maxgi|9205433 Length: 605 TBLASTN contig gi|12488109 Identities: 0.614Score: 592 from gi|12773915 Positives: 0.733 Probability: 1.500000e−57soybean gi|15286042 Frames: 2 gi|18731515 consensus: SEQ ID NO: 11 OneEST Triticum gi|9742370 Length: 1569 TBLASTN contig aestivum gi|20309688Identities: 0.638 Score: 992 from gi|25194283 Positives: 0.745Probability: 4.000000e−100 wheat gi|25235692 Frames: 1 gi|25237006gi|25243036 gi|25261519 gi|25280706 gi|25431065 gi|25547029 gi|25560875gi|32546080 gi|32670437 consensus: SEQ ID NO: 13 One EST Oryza sativagi|5456487 Length: 463 TBLASTN contig gi|13420908 Identities: 0.766Score: 469 from rice gi|700187 Positives: 0.901 Probability:9.800000e−45 consensus: SEQ Frames: 1 ID NO: 9 One EST Populusgi|3857688 Length: 1262 TBLASTN contig tremula gi|23959842 Identities:0.674 Score: 1040 from gi|23960044 Positives: 0.774 Probability:9.900000e−106 poplar gi|23963690 Frames: 2 gi|23997967 gi|23998879gi|24062476 gi|24065210 gi|28609230 consensus: SEQ ID NO: 10 One ESTLycopersicon gi|5896833 Length: 927 TBLASTN contig esculentum gi|5896833Identities: 0.652 Score: 949 from gi|5896833 Positives: 0.769Probability: 3.600000e−106 tomato gi|12628275 Frames: 2 gi|12628275gi|12628275 gi|16242572 consensus: SEQ ID NO: 8 One EST Zea maysgi|5739680 Length: 864 TBLASTN contig gi|21208932 Identities: 0.626Score: 450 from gi|407242 Positives: 0.772 Probability: 3.000000e−50corn gi|407243 Frames: 3 consensus: SEQ ID NO: 12 One EST Gossypiumgi|3326250 Length: 681 TBLASTN contig hirsutum Identities: 0.629 Score:415 from Positives: 0.750 Probability: 8.600000e−40 cotton Frames: 1

Closest Plant Homologs: At1g73650 Arabidopsis gi|18410409 Identities:0.893 BLASTP thaliana Positives: 0.893 Score: 1351 Frames: N P =3.700000e−137 At1g18180 Arabidopsis gi|15221003 Identities: 0.733 BLASTPthaliana Positives: 0.806 Score: 1122 Frames: N P = 6.900000e−113gi|13937298 Oryza sativa gi|13937298 Identities: 0.641 BLASTP Positives:0.760 Score: 1004 Frames: N P = 2.200000e−100 gi|31213708 Anophelesgi|31213708 Identities: 0.415 BLASTP gambiae gi|21299548 Positives:0.588 Score: 592 Frames: N P = 1.000000e−56 gi|21355723 Drosophilagi|21355723 Identities: 0.394 BLASTP melanogaster gi|28574404 Positives:0.564 Score: 555 gi|16648114 Frames: N P = 8.300000e−53 gi|23093921gi|28380570 gi|38105747 Magnaporthe gi|38105747 Identities: 0.416 BLASTPgrisea70-15 Positives: 0.549 Score: 509 Frames: N P = 6.300000e−48gi|43807390 environmenta gi|43807390 Identities: 0.336 BLASTP 1 sequencePositives: 0.529 Score: 418 Frames: N P = 2.700000e−38

This NCBI entry for At1g73650 (NP_(—)849882.1) is a predictedtrans-membrane protein. There are six predicted trans-membrane domains(predicted by TMHMM; amino acid residues 9-31; 64-86; 99-118; 138-155;185-204; 209-231). The first trans-membrane domain overlaps with thesignal anchor sequence (predicted by SignalP; probability=0.990; aminoacid residues 1-28).

Psort2 predicts that At1g73650 may be localized to the endoplasmicreticulum, plasma membrane or the mitochondria (32% endoplasmicreticulum, 28% plasma membrane, 24% mitochondrial, 4% nuclear, 4% golgiapparatus, 4% vacuolar, 4% vesicles of secretory system by Psort2).However, At1g73650 is unlikely to be targeted to the mitochondria basedon TargetP prediction.

Pfam analysis predicts that At1g73650 has one domain of unknown function(PF06966, amino acid residues 24-252): Model Domain seq-f* seq-t hmm-fhmm-t score E-value PF06966 1/1 24 252 .. 1 266 [ ] 453.0 4.9e−133*Seq-f refers to “sequence-from” and seq-t refers to “sequence-to.” Thetwo periods following the seq-t number indicate that the matching regionwas within the sequence and did not extend to either end. The twobrackets indicate that the match spanned the entire length of theprofile HMM. hmm-f and hmm-t refer to the beginning and endingcoordinates of the matching portion of the profile HMM.

Example 5

Transformed explants of rapeseed, soy, corn, sunflower, cotton, cocoa,safflower, oil palm, coconut palm, flax, castor and peanut are obtainedthrough Agrobacterium tumefaciens-mediated transformation ormicroparticle bombardment. Plants are regenerated from transformedtissue. The greenhouse grown plants are then analyzed for the gene ofinterest expression levels as well as oil levels.

Example 6

This example provides analytical procedures to determine oil and proteincontent, mass differences, amino acid composition, free amino acidlevels, and micronutrient content of transgenic maize plants.

Oil levels (on a mass basis and as a percent of tissue weight) of firstgeneration single corn kernels and dissected germ and endosperm aredetermined by low-resolution 1H nuclear magnetic resonance (NMR) (Tiwariet al., JAOCS, 51:104-109 (1974); or Rubel, JAOCS, 71:1057-1062 (1994)),whereby NMR relaxation times of single kernel samples are measured, andoil levels are calculated based on regression analysis using a standardcurve generated from analysis of corn kernels with varying oil levels asdetermined gravimetrically following accelerated solvent extraction.One-way analysis of variance and the Student's T-test (JMP, version4.04, SAS Institute Inc., Cary, N.C., USA) are performed to identifysignificant differences between transgenic and non-transgenic kernels asdetermined by transgene-specific PCR.

Oil levels and protein levels in second generation seed are determinedby NIT spectroscopy, whereby NIT spectra of pooled seed samplesharvested from individual plants are measured, and oil and proteinlevels are calculated based on regression analysis using a standardcurve generated from analysis of corn kernels with varying oil orprotein levels, as determined gravimetrically following acceleratedsolvent extraction or elemental (% N) analysis, respectively. One-wayanalysis of variance and the Student's T-test are performed to identifysignificant differences in oil (% kernel weight) and protein (% kernelweight) between seed from marker positive and marker negative plants.

The levels of free amino acids are analyzed from each of the transgenicevents using the following procedure. Seeds from each of the transgenicplants are crushed individually into a fine powder and approximately 50mg of the resulting powder is transferred to a pre-weighed centrifugetube. The exact sample weight is recorded and 1.0 ml of 5%trichloroacetic acid is added to each sample tube. The samples are mixedat room temperature by vortex and then centrifuged for 15 minutes at14,000 rpm on an Eppendorf microcentrifuge (Model 5415C, BrinkmannInstrument, Westbury, N.Y.). An aliquot of the supernatant is removedand analyzed by HPLC (Agilent 1100) using the procedure set forth inAgilent Technical Publication “Amino Acid Analysis Using the ZorbaxEclipse-AAA Columns and the Agilent 1100 HPLC,” Mar. 17, 2000.

Quantitative determination of total amino acids from corn is performedby the following method. Kernels are ground and approximately 60 mg ofthe resulting meal is acid-hydrolyzed using 6 N HCI under reflux at 100°C. for 24 hrs. Samples are dried and reconstituted in 0.1 N HCI followedby precolumn derivatization with α-phthalaldehyde (OPA0 for HPLCanalysis. The amino acids are separated by a reverse-phase ZorbaxEclipse XDB-C18 HPLC column on an Agilent 1100 HPLC (Agilent, Palo Alto,Calif.). The amino acids are detected by fluorescence. Cysteine,proline, asparagine, glutamine, and tryptophan are not included in thisamino acid screen (Henderson et al., “Rapid, Accurate, Sensitive andReproducible HPLC Analysis of Amino acids, Amino Acid Analysis UsingZorbax Eclipse-AAA Columns and the Agilent 1100 HPLC,” AgilentPublication (2000); see, also, “Measurement of Acid-Stable Amino Acids,”AACC Method 07-01 (American Association of Cereal Chemists, ApprovedMethods, 9th edition (LCCC# 95-75308)). Total tryptophan is measured incorn kernels using an alkaline hydrolysis method as described (ApprovedMethods of the American Association of Cereal Chemists-10^(th) edition,AACC ed, (2000) 07-20 Measurement of Tryptophan-Alakline Hydrolysis).

Tocopherol and tocotrienol levels in seeds are assayed by methodswell-known in the art. Briefly, 10 mg of seed tissue are added to 1 g ofmicrobeads (Biospec Product Inc, Barlesville, Okla.) in a sterilemicrofuge tube to which 500 μl 1% pyrogallol (Sigma Chemical Co., St.Louis, Mo.)/ethanol have been added. The mixture is shaken for 3 minutesin a mini Beadbeater (Biospec) on “fast” speed, then filtered through a0.2 μm filter into an autosampler tube. The filtered extracts areanalyzed by HPLC using a Zorbax silica HPLC column (4.6 mm×250 mm) witha fluorescent detection, an excitation at 290 nm, an emission at 336 nm,and bandpass and slits. Solvent composition and running conditions areas listed below with solvent A as hexane and solvent B as methyl-t-butylether. The injection volume is 20 μl, the flow rate is 1.5 ml/minute andthe run time is 12 minutes at 40° C. The solvent gradient is 90% solventA, 10% solvent B for 10 minutes; 25% solvent A, 75% solvent B for 11minutes; and 90% solvent A, 10% solvent B for 12 minutes. Tocopherolstandards in 1% pyrogallol/ethanol are run for comparison (α-tocopherol,γ-tocopherol, β-tocopherol, δ-tocopherol, and tocopherol (tocol)).Standard curves for alpha, beta, delta, and gamma tocopherol arecalculated using Chemstation software (Hewlett Packard). Tocotrienolstandards in 1% pyrogallol/ethanol are run for comparison(α-tocotrienol, γ-tocotrienol, β-tocotrienol, δ-tocotrienol). Standardcurves for α-, β-, δ-, and γ-tocotrienol are calculated usingChemstation software (Hewlett Packard).

Carotenoid levels within transgenic corn kernels are determined by astandard protocol (Craft, Meth. Enzymol., 213:185-205 (1992)).Plastiquinols and phylloquinones are determined by standard protocols(Threlfall et al., Methods in Enzymology, XVIII, part C, 369-396 (1971);and Ramadan et al., Eur. Food Res. Technol., 214(6):521-527 (2002)).

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1. A transgenic plant comprising a plant transformation vectorcomprising a nucleotide sequence that encodes or is complementary to asequence that encodes a HIO1002 polypeptide comprising the amino acidsequence of SEQ ID NO:2, 4, or 6, or an ortholog thereof, whereby thetransgenic plant has a high oil phenotype relative to control plants. 2.The transgenic plant of claim 1, which is selected from the groupconsisting of rapeseed, soy, corn, sunflower, cotton, cocoa, safflower,oil palm, coconut palm, flax, castor and peanut.
 3. A plant partobtained from the plant according to claim
 1. 4. The plant part of claim3, which is a seed.
 5. Meal, feed, or food produced from the seed ofclaim
 4. 6. A method of producing oil comprising growing the transgenicplant of claim 1 and recovering oil from said plant.
 7. The method ofclaim 6, wherein the oil is recovered from a seed of the plant.
 8. Amethod of producing a high oil phenotype in a plant, said methodcomprising: a) introducing into progenitor cells of the plant a planttransformation vector comprising a nucleotide sequence that encodes oris complementary to a sequence that encodes a HIO1002 polypeptidecomprising the amino acid sequence of SEQ ID NO:2, 4, or 6, or anortholog thereof, and b) growing the transformed progenitor cells toproduce a transgenic plant, wherein said polynucleotide sequence isexpressed, and said transgenic plant exhibits an altered oil contentphenotype relative to control plants.
 9. A plant obtained by a method ofclaim
 8. 10. The plant of claim 9, which is selected from the groupconsisting of rapeseed, soy, corn, sunflower, cotton, cocoa, safflower,oil palm, coconut palm, flax, castor and peanut.
 11. A method ofgenerating a plant having a high oil phenotype comprising identifying aplant that has an allele in its HIO1002 gene that results in increasedoil content compared to plants lacking the allele and generating progenyof said identified plant, wherein the generated progeny inherit theallele and have the high oil phenotype.
 12. The method of claim 11 thatemploys candidate gene/QTL methodology.
 13. The method of claim 11 thatemploys TILLING methodology.
 14. A feed, meal, grain, or seed comprisinga polypeptide encoded by the nucleic acid sequence as set forth in SEQID NO: 1, 3 or
 5. 15. A feed, meal, grain, or seed comprising apolypeptide comprising an amino acid sequence as set forth in SEQ IDNO:2, 4, or 6, or an ortholog thereof.